U.S. patent application number 10/088728 was filed with the patent office on 2003-09-04 for a method for preparing massively rmlc protein and purified rmlc protein of mycobacterium tuberculosis by it.
Invention is credited to Lee, Tae-Yoon.
Application Number | 20030166234 10/088728 |
Document ID | / |
Family ID | 26638241 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166234 |
Kind Code |
A1 |
Lee, Tae-Yoon |
September 4, 2003 |
A METHOD FOR PREPARING MASSIVELY RmlC PROTEIN AND PURIFIED RmlC
PROTEIN OF MYCOBACTERIUM TUBERCULOSIS BY IT
Abstract
This invention relates to a mass preparation method of RmlC
protein and the purified RmlC protein by the said method. RmlC is a
gene product of rmlC, which is one of the biosynthesis genes of
rhamnose that is an important element that consists cell wall of
Mycobacterium tuberculosis. To be more specific, this invention
improves the following disadvantage of recombinant RmlC protein of
Mycobacterium tuberculosis previously reported that contains
unnecessary 15 amino acids into the natural RmlC protein. This
invention relates to a recombinant plasmid, a recombinant E. coli
that is transformed by the plasmid, preparation method of
Mycobacterium tuberculosis RmlC recombinant protein using the
recombinant E. coli, purification method of recombinant RmlC
protein, and the RmlC protein purified by the said method.
Inventors: |
Lee, Tae-Yoon; (Taegu-shi,
KR) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
26638241 |
Appl. No.: |
10/088728 |
Filed: |
March 22, 2002 |
PCT Filed: |
July 23, 2001 |
PCT NO: |
PCT/KR01/01251 |
Current U.S.
Class: |
435/200 ;
435/252.33; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 15/70 20130101 |
Class at
Publication: |
435/200 ;
435/69.1; 435/252.33; 536/23.2; 435/320.1 |
International
Class: |
C12N 009/24; C07H
021/04; C12N 015/74; C12P 021/02; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 22, 2000 |
KR |
2000/42274 |
Nov 8, 2000 |
KR |
2000/66236 |
Claims
1. Plasmid pRMC609 that contains Rhamnose biosynthesis gene rmlC of
Mycobacterium tuberculosis on general expression vector pET23b
2. E. coli that is transformed with the plasmid pRMC609
3. A transformant (KCCM-10192) of claim 2, wherein the host strain
is E. coli BL21 (PlysS)
4. A manufacturing method of recombinant RmlC protein comprising
steps of: culturing the transformant of claim 2; and inducing
expression of Rhamnose biosynthesis gene rmlC of Mycobacterium
tuberculosis.
5. A purification method of recombinant RmlC protein comprising
steps of: (i) culturing the transformant of claim 2 and inducing
expression of Rhamnose biosynthesis RmlC protein; (ii) lysing the
transformant the expression of which is induced in the step (i) and
obtaining and anion-exchange chromatographing the supernatant;
(iii) collecting the fractions of the step (ii) that contain
recombinant RmlC protein and obtaining the supernatant by warming
the fraction between 50-80.degree. C.; and (iv) FPLC gel-filtration
chromatographing the supernatant obtained in the step (iii).
6. Rhamnose biosynthesis RmlC protein of Mycobacterium tuberculosis
purified by the purification method of RmlC protein of claim 5.
Description
TECHNICAL FIELD
[0001] This invention relates to a mass preparation method of RmlC
protein, a gene product of rmlC that is one of the genes
synthesizing rhamnose, which is an important element that
constitutes cell wall of Mycobacterium tuberculosis. To be more
specific, this invention relates to an improvement of disadvantages
such as expression efficiency reduction of RmlC protein for the
development of anti-tuberculosis drug(s) because of the fusion of
15 unnecessary amino acids into Mycobacterium tuberculosis RmlC
recombinant protein as reported previously (Stern R. J. et al.
Microbiology 145:663-671(1999)), problems of crystal formation
because of the fusion of unnecessary amino acids when crystal of
RmlC protein is formed, and extension of required time because of
the fusion of unnecessary amino acids when the structure of RmlC
protein is determined by X-ray crystallography. This method
contains a recombinant plasmid that expresses RmlC protein itself
without unnecessary amino acids and a method that can produce RmlC
protein of Mycobacterium tuberculosis in large quantities that
contains enzymatic activity of the corresponding RmlC protein, a
recombinant E. coli transformed by the plasmid, a preparation
method of Mycobacterium tuberculosis RmlC recombinant protein using
E. coli, a purification method of recombinant RmlC protein prepared
by said method, and the RmlC protein purified by said method.
BACKGROUND ART
[0002] Approximately 1.7 billion people, 32% of the world
population, are infected by tuberculosis. There are 8 million new
patients every year and about 34% of them, 2.7 million, died from
this serious disease. In Korea, it is estimated that there are
approximately 0.7 million tuberculosis patients, 0.14 million new
patients every year, and 4,000 people died from the disease.
Therefore, tuberculosis remains as a serious health problem.
[0003] Tuberculosis is a chronic infectious disease caused by
Mycobacterium tuberculosis and currently the prevalence is
increasing worldwide as a complication, of acquired
immunodeficiency syndrome(AIDS). Also, many of the recent
Mycobacterium tuberculosis has multiple-drug resistance that makes
tuberculosis treatment more difficult.
[0004] Therefore, to eradicate tuberculosis, various efforts are
needed such as development of a rapid diagnostic technique,
development of a new drug, molecular epidemiological study and also
study on vaccine development. Among those, development of a new
drug is one of the most urgent for the purpose.
[0005] The main classical method for the antibiotics development
was random screening of an antibacterial molecule(s) from
microorganisms or natural materials. However, this approach has
disadvantage such as much time and investment.
[0006] Recently, as a more advanced approach, new drug development
is being performed. This approach includes the study of metabolic
processes of corresponding microorganism and targeting an essential
enzyme for its survival. It is desirable that the target enzyme is
specific to the corresponding microorganism and does not exist in
the cell of mammalians including human being. However, this
approach has similar disadvantages with the previously described
method in terms that it needs screening of materials that inhibit
the target enzyme activity.
[0007] As a method to overcome these problems, Kuntz et al.
proposed the concept of "structure-based drug design" in early
1990. This is a strategy to design and synthesize an inhibitor of a
target enzyme by determining the structure of a target enzyme. The
time required for the development of a new drug can be
substantially shortened by this approach. This approach needs
understanding of metabolic pathway that a target enzyme involves,
cloning of the corresponding gene, overexpression, and purification
in the first place. The purified enzyme can be used for crystal
formation and structure determination by X-ray crystallography.
Therefore, the 4-step procedure below is required to develop a new
drug for tuberculosis based on the concept explained above.
[0008] First, it needs cloning of target enzyme gene,
overexpression, purification, and establishment of activity
detection method of the corresponding enzyme.
[0009] Second, it needs crystal formation, structural analysis of
the purified target enzyme, and design and synthesis of enzyme
inhibitors.
[0010] Third, it needs demonstration of inhibition effect of enzyme
activity of the inhibitors designed and synthesized and selection
of new drug candidates through evaluation of the bactericidal
activity against Mycobacterium tuberculosis.
[0011] Fourth, it needs animal test and clinical test of new drug
candidates selected.
[0012] The First thing for the development of new anti-tuberculosis
drugs according this new concept is to screen components that are
involved in the pathogenicity of or vital to Mycobacterium
tuberculosis and the corresponding genes.
[0013] One of the factors that contribute to the pathogenicity of
Mycobacterium tuberculosis is its thick cell wall. It contributes
to antibiotics resistance and functions as a permeability barrier.
So far, a lot of information has been accumulated about the cell
wall structure of acid-fast bacteria where Mycobacterium
tuberculosis belongs. The cell wall of Mycobacterium tuberculosis
has a basic structure that includes two large polymers named
peptidoglycan and arabinogalactan. These two polymers are linked by
a covalent binding. Ethainbutol, currently being used as an
anti-tuberculosis drug, is known to exhibit anti-tuberculosis
activity by inhibition of arabinogalactan synthesis in
Mycobacterium tuberculosis. Meanwhile, the base unit of wax
structure, which contributes to the hydrophobicity of cell wall of
Mycobacterium tuberculosis, is known to be mycolic acid. The
mycolic acid is linked to arabinogalactan through covalent binding
and arabinogalactan is again linked to peptidoglycan layer.
According to a molecular structural study on the cell wall of
Mycobacterium tuberculosis, there is a very small bridge-like
structure between arabinogalactan and peptidoglycan. Thus these two
polymers are connected to each other by this structure. In more
detail, galactan part of the arabinogalactan and muramyl residue of
peptidoglycan are linked by the bridge structure of
[L-Rhamnose-N-Acetylglucosamine-Phosphate] (Reference: FIG. 1).
Therefore, if the biosynthesis of either Rhamnose or
N-acetylglucosamine that are the components of this bridging
structure is blocked, cell wall of Mycobacterium tuberculosis gets
weakened and, as a result, the material that has this effect could
be a new drug candidate for tuberculosis treatment.
[0014] Among the components of this bridge structure Rhamnose is
one of the components of O antigen, a component of
lipopolysaccharide that is existed in cell wall of enteric
bacteria. Numerous studies have been performed about the
biosynthesis of Rhamnose in enteric bacteria. According to these
studies, Rhamnose is synthesized as a form of deoxythymidine
diphosphate(dTDP)-Rhamnose, and dTDP-Rhamnose is known to be the
molecule providing Rhamnose to the cell wall of both Mycobacterium
tuberculosis and general bacteria. These studies showed
biosynthesis pathway of dTDP-Rhamnose in enteric bacteria,
dTDP-Rhamnose is synthesized from deoxythymidine triphosphate(TTP)
and glucose-1-phosphate, and 4 genes of rmlA, rmlB, rmlC and rmlD
are involved. RmlA protein is glucose-1-phosphate
thymidylyltransferase that produces dTDP-glucose from
.alpha.-D-glucose-1-phosphate and dTTP. RmlB protein is
dITDP-4,6-dehydratase that produces dTDP-4-keto-6-deoxyglucose from
dTDP-glucose. RmlC protein is dTDP-6-deoxyglucose-3,5-epimerase
that produces dTDP-4-keto-6-deoxymannose from
dTDP-4-keto-6-deoxyglueose. RmlD protein is dTDP-6-deoxymannose
dehydrogenase that produces dTDP-Rhamnose from
dTDP-4-keto-6-deoxymannose. (Reference: FIG. 2)
[0015] In this respect, studies on genes and proteins that are
involved in biosynthesis of dTDP-Rhamnose are very important as
targets for the new drug development against tuberculosis.
Currently, studies on new drug development targeting the
dTDP-Rhamnose synthesis are in its very early stage in
Mycobacterium tuberculosis. This is due to lack of information on
metabolic pathways to synthesize dTDP-Rhamnose as well as
information on enzyme and corresponding genes.
[0016] Meanwhile, it is ideal that only a single copy of the drug
target gene or enzyme exists in the corresponding organism.
According to the whole nucleotide sequence information of
Mycobacterium tuberculosis standard isolate H37Rv that are revealed
in 1998, rmlA and rmlB have more than two copies while rmlC and
rmlD have only one copy. Therefore, it showed that the target genes
for the new drug development through inhibition of dTDP-Rhamnose
biosynthesis are mostly likely to be rmlC and rmlD. (Reference:
Cole S. T. et al. Nature, 393:537-544(1998))
DISCLOSURE OF THE INVENTION
[0017] As a basic step to develop a new drug against tuberculosis
that inhibits dTDP-Rhamnose, a target molecule, the inventor made
an effort to express rmlC in large quantities. As a result, the
inventor confirmed that E. coli transformed with a recombinant
plasmid that contains rmlC gene of standard Mycobacterium
tuberculosis H37Rv produces RmlC protein of its natural state
without the fusion of unnecessary amino acids as well as that
corresponding RmlC protein contained its enzymatic activity.
[0018] After all, the first objective of this invention is to
provide a recombinant plasmid that can produce RmlC protein of
Mycobacterium tuberculosis in large quantities.
[0019] The second objective of this invention is to provide
recombinant E. coli that is transformed by the said plasmid.
[0020] The third objective of this invention is to provide a
preparation method of recombinant RmlC using the said E. coli.
[0021] The fourth objective of this invention is to provide
purification method of the recombinant RmlC protein that is
produced as described above.
[0022] The fifth objective of this invention is to provide purified
RmlC protein using the said purification method of recombinant RmlC
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a supplementary picture describing the structure
of cell wall of Mycobacterium tuberculosis.
[0024] FIG. 2 describes the biosynthesis pathway of Rhamnose.
[0025] FIG. 3 is a picture of agarose gel electrophoresis
presenting 609 bp rmlC gene that are amplified by PCR with genome
DNA of standard Mycobacterium tuberculosis H37Rv as a template.
[0026] FIG. 4 is a picture of SDS-PAGE electrophoresis presenting
purification of RmlC protein from E. coli that is transformed with
recombinant plasmid pRMC609.
[0027] FIG. 5 is a chromatogram presenting the result of enzymatic
activity measurement of the RmlC protein using High Performance
Liquid Chromatography (HPLC)
BEST MODE FOR CARRYING OUT THE INVENTION
[0028] The following is a more detailed explanation of the present
invention.
[0029] In this invention, for the preparation of the recombinant
protein RmlC, genomic DNA is prepared from H37Rv first. Then the
rmlC gene is amplified by PCR using this genomic DNA as a template.
A recombinant plasmid is constructed that expresses RmlC protein in
large quantities by cloning the amplified rmlC gene on pET23b, a
gene overexpression plasmid. An E. coli is transformed by this
recombinant plasmid and the recombinant RmlC protein of
Mycobacterium tuberculosis is prepared from the transformant in
large quantities through the process of cultivation and
induction.
[0030] Meanwhile, to separate RmlC protein expressed from the said
transformant, the protein expression is induced by cultivation of
the transformant and the supernatant of the transformant protein is
fractionated by anion exchange resin chromatography. The fraction
that contains RmlC protein is warmed between 50.degree. C. and
80.degree. C., ideally at 65.degree. C., and the supernatant
obtained is fractionated by FPLC gel-filtration chromatography.
[0031] Next, the size of the purified recombinant RmlC protein on
an electrophoresis gel is confirmed by calculating the estimated
protein size deduced from the nucleotide sequence of the rmlC gene.
The enzymatic activity of the recombinant RmlC protein is examined
by HPLC whether it retains its natural state. As a result, the RmlC
protein prepared and purified by the said method in this invention
does not have unnecessary amino acids and exhibits enzymatic
activity of RmlC itself. The size of recombinant RmlC protein
comprised of the same amino acid sequences as those of RmlC in its
natural state is smaller than the previously reported recombinant
RmlC protein that has 15 additional unnecessary amino acids.
Therefore, it is clear that the efficiency of mass preparation and
purification of smaller RmlC is greater than those of large RmlC
protein when identical or similar mass preparation and purification
methods are applied. Also, purified RmlC protein that are prepared
and purified by this method is closer to Mycobacterium tuberculosis
RmlC protein of its natural state. Thus, higher accuracy is
expected when it is used for crystal formation and third
dimensional structure determination by X-ray crystallography.
[0032] The following will be more detailed explanation of the
present invention by examples.
[0033] These examples are only to explain the present invention and
embodiments of the present invention are not limited only to the
above, and it is evident that it can be diversely modified by a
person who has ordinary knowledge in the appropriate field, within
the technical idea of the present.
EXAMPLE 1
Culture of Mycobacterium tuberculosis
[0034] One loop amount of standard Mycobacterium tuberculosis
isolate H37Rv, that has been cultured and stored in Ogawa solid
medium, is inoculated to Middlebrook 7H9 liquid medium (Difco, Co.
USA) with ADC (Albumin fraction V: bovine, Dextrose and Catalase:
Difco, Co. USA). The culture is incubated for more than 4 weeks
with light shaking (about 100 rpm) at 35-37.degree. C. until enough
bacteria can be obtained. Middlebrook 7H9 liquid medium is made by
dissolving 4.7 g of medium powder in 900 mL of distilled water
followed by autoclave. After autoclave, 2 mL of glycerol, that is
autoclaved separately, and 10 mL of Middlebrook ADC is added.
[0035] The bacteria are obtained through centrifugation of the
culture at 3000.times.g for 20 minutes. A portion of the bacteria
is kept at -70.degree. C. for storage after addition of Brucella
broth and 15% glycerol. The rest of the bacteria portion is kept at
-20.degree. C. and is used for various experiments.
EXAMPLE 2
Genomic DNA Separation from Mycobacterium tuberculosis Cultured in
Large Quantities
[0036] Mycobacterium tuberculosis cultured in large quantities from
example 1 is collected and used for DNA preparation after
incubating at 75.degree. C. for 20 minutes for virulence
attenuation. First, the bacteria are frozen at -70.degree. C. and
thawed. Then it was left at 37.degree. C. for 1 hour in the
presence of 2 mg/mL of lysozyme. Then it was left at 55.degree. C.
for 48 hours in the presence of 1% SDS and 1 mg/mL of proteinase K.
The lysate is washed twice at 55.degree. C. for 30 minutes with TE
buffer solution that contains 0.04 mg/mL of
Phenylmethylsulfonylfluoride, an inhibitor of proteinase K. Then,
equal amount of chloroform-isoamylalcohol mixture
(chloroform:isoamylalcohol=24- :1(v/v)) is added, mixed well and
centrifuged. The upper liquid portion is taken and the genomic DNA
is precipitated by adding 0.6 times volume of isopropanol.
Example 3
Amplification of Mycobacterium tuberculosis rmlC Gene by PCR
[0037] Mycobacterium tuberculosis rmlC gene is amplified by PCR
using synthesized primer set p1/p2 (p1: sequence number 1; p2:
sequence number 2) with genome DNA of Mycobacterium tuberculosis
prepared in example 2 as a template.
[0038] As seen in sequence number 1 and 2, there are EcoRI (GAATTC)
and NdeI (CATATG) cut-off regions that contain initiation codon
(ATG) at 5'-terminus of p1. There are BamHI cut-off region (GGATCC)
and CTA sequence that is complementary base sequence to termination
codon (TGA). EcoRI, NdeI, and BamHI cut-off regions in p1 and p2
are for the effective cloning of PCR product to the plasmid after
PCR.
[0039] PCR reaction mixture contains 20 mM Tris-HCl (pH 8.8), 10 mM
KCl, 2 mM (NH.sub.4).sub.2SO.sub.4, 0.1% Triton X-100, 0.5 .mu.M of
each primer, 200 .mu.M of dNTPs, and 2.5 unit of Vent DNA
Polymerase (New England Biolabs, Inc., USA). PCR is performed after
1 ng of template DNA is added to the reaction mixture with a total
volume of 50 .mu.l. To prevent water evaporation during the
reaction, mineral oil was dropped on the reaction mixture. PCR
temperature cycle contains once for 5 minutes at 94.degree. C.; 30
seconds at 96.degree. C., 30 seconds at 60.degree. C., and 30
seconds at 72.degree. C.(25 times); and once for 7 minutes at
72.degree. C.
[0040] FIG. 3 is the picture of agarose gel electrophoresis showing
the 609 bp rmlC gene amplified by PCR. In FIG. 3, M is 100 bp
ladder DNA and is a standard DNA for estimating the size of DNA. As
seen in FIG. 3, the expected 609 bp band was observed.
Example 4
Mass Expression and Purification of Mycobacterium tuberculosis RmlC
protein
[0041] The rmlC gene amplified in Example 3 does not have errors
during DNA synthesis, which can be occurred during PCR reaction.
Amplified rmlC gene is digested with restriction enzyme NdeI and
BamHI, and is cloned to pET23b (Novagen Co., USA), a mass
expression vector, and the recombinant plasmid is named
pRMC609.
[0042] Plasmid pRMC609 is introduced to BL21 (PlysS) E. coli, a
host strain of expression vector pET23b. The E. coli BL21 (PlysS)
that are transformed with pRMC609 is named BRMC609, and deposited
to Korea Culture Center of Microorganisms (KCCM) on Jul. 3, 2000
(Deposit Number KCCM-10192).
[0043] BRMC609 transformant is cultured in 3 liters of LB liquid
medium that contains 50 .mu.g/mL of ampicillin and the expression
of the protein is induced at 37.degree. C. for 4 hours by adding
0.5 mM IPTG (Isopropylthiogalactoside) at late logarithmic
phase.
[0044] Then E. coli cell is obtained by centrifuge and recombinant
RmlC protein is prepared and purified using the chromatography
method as follows. That is, to E. coli cells obtained, 1 mM EDTA,
0.1 mM PMSF, 1 .mu.g/mL leupeptin, 0.1 mg/mL lysozyme, and 200 mL
of 20 mM Tris-HCl buffer solution (pH 7.5) that contains 1 .mu.g/ml
DNase are added, and the bacteria is dissolved after repeated
freezing and thawing. After centrifugation at 15,000 rpm for 20
minutes, the supernatant is placed in a DEAE Sepharose CL 6B column
that is equilibrated by 20 mM Tris-HCl buffer solution (pH 7.5)
containing 2 mM EDTA. The protein is eluted with a straight-line
concentration gradient of NaCl of 0 to 0.5M that is dissolved in 10
mM Tris-HCl buffer solution (pH 7.5). The fractions that contain
RmlC protein are checked by Coomassie blue staining after
SDS-PAGE.
[0045] After collecting fractions that contains recombinant RmlC
protein, the purity of the protein is increased using its heat
stability. The RmlC protein solution is centrifuged at 12,000 rpm
for 20 minutes after incubation in 65.degree. C. water bath for 10
minutes, and the liquid is concentrated using Centriprep 10
concentrator (Amicon Co., USA)
[0046] The final purification is FPLC gel-filtration
chromatography. The Superdex column is equilibrated with 20 mM
Tris-HCl buffer solution (pH 7.5) that contains 0.1 M NaCl and
0.02% sodium azide. The fractions that contain RmlC protein are
examined by Coomassie blue staining after SDS-PAGE. After
collecting fractions that contain recombinant RmlC protein, the
protein is finally concentrated using Centriprep 10 concentrator
(Amicon Co., USA). As a result, the 22.3 kDa protein is confirmed
to be purified as estimated from the nucleotide sequence of rmlC
gene. (Reference: FIG. 4)
Example 5
Measurement of Enzymatic Activity of RmlC Protein Using High
Performance Liquid Chromatography
[0047] Enzymatic activity of the purified RmlC protein in example 4
is examined by High Performance Liquid Chromatography (HPLC). In
the analyzing mixture, 2 mL of dTDP-Glucose, 6 nmol of NADPH, 50
.mu.g of crude soluble protein obtained from E. coli BW24970 (RmlB
and RmlD proteins that are involved in making dTDP-Rhamnose from
dTDP glucose are contained here), 2 .mu.g of purified RmlC protein
and 1 nM, of MgCl.sub.2 are dissolved in 50 mM Hepes buffer
solution (pH 7.6). After this mixture solution is incubated for 1
hour at 37.degree. C., 67 .mu.L of ethanol is added. Denatured
protein is removed by centrifugation at 14,000.times.g for 10
minutes, and the supernatant is placed in Dionex PA-100 HPLC column
(Dionex Co., USA). Then the column is eluted by 75 mM
KH.sub.2PO.sub.4 and absorbance at 254 nm is measured. Synthesis of
dTDP-Rhamnose can be confirmed using dTDP-glucose and dTDP-Rhamnose
as standards. The result shows the synthesis of dTDP-Rhamnose only
in the reaction where purified RmlC protein was added not in the
reaction where RmlC protein was not added. (Reference: FIG. 5)
[0048] As explained and confirmed above, according to the present
invention, the RmlC protein expressed in large quantities from a
recombinant plasmid (this contains Rhamnose biosynthesis gene rmlC,
a known target for new drug development against Mycobacterium
tuberculosis) is an RmlC protein of its natural state without the
fusion of unnecessary amino acids and retains the enzymatic
activity of corresponding protein. Therefore, recombinant plasmid,
its transformant, preparation and purification method of the
recombinant RmlC protein presented in this invention is very
effective in mass preparation of Rhamnose biosynthesis enzyme RmlC,
and the RmlC protein that is prepared in large quantities and
purified using said method can be very useful in protein crystal
formation and three dimensional structural determination of RmlC
protein by X-ray crystallography that are necessary steps for
structure-based new drug development.
Sequence CWU 1
1
2 1 34 DNA Artificial Sequence Single stranded oligonucleotide
primer 1 ggaattccat atgaaagcac gcgaactcga cgtc 34 2 28 DNA
Artificial Sequence Single stranded oligonucleotide primer 2
cgggatccta ggtgccgcgc atctcccc 28
* * * * *